The mechanisms responsible for the development of asthma in atopic patients include genetic predisposition and the effects of environmental exposures to inflammatory stimuli in the airways of susceptible individuals (Bleecker, E. R., and D. A. Meyers in Genetics of Allergy and Asthma. M. N. Blumenthal, and B. Bjorksten, (eds. Marcel Dekker, New York, p. 307 1997). Asthma represents a chronic inflammatory process of the airways. The consequences of chronic inflammation in the asthmatic airways include increased numbers of fibroblasts and the deposition of extracellular matrix (ECM) such as collagen, fibronectin, and laminin within the airway wall (Altraja, A., et al. Am. J. Respir. Cell. Mol. Biol. 15: 482, 1996; Roche, W. R., et al. Lancet. 1:520, 1989). The plasminogen activator (PA) system has an important role in controlling endogenous fibrosis and regulating ECM proteolysis relevant to tissue remodeling (Gabazza, E. C., et al. Lung. 177: 253, 1999). The tissue-type PA (tPA) and urokinase-type PA (uPA) converts plasminogen to plasmin, which enhances proteolytic degradation of the ECM. An important mechanism in the regulation of PA activity is inhibition of uPA or tPA by three major inhibitors, which are PAI-1, PAI-2, and PAI-3 (Kruitoff, E. K. Enzyme 40: 113, 1988). Among these three inhibitors, PAI-1 is the most important in controlling lung fibrosis (Geiger, M., et al, Immunopharmacology 32: 53, 1996; Lardot, C., et al. Eur. Respir. J. 11: 912, 1988; Kruitoff E. K., et al. J. Biol. Chem. 261: 11207, 1986). PAI-1 overexpressing mice suffered severe lung injury and deposition of ECM after bleomycin challenge (Eitzman, D. T., et al. J. Clin. Invest. 97: 232, 1996) or hyperoxia (Barazzone, C., et al. J. Clin. Invest. 98: 2666, 1996), whereas PAI-1 deficient mice were protected against such a fibrotic reaction. These findings show that PAI-1 is closely associated with fibrosis and ECM accumulation after lung injury or inflammation. Recently, the induction of PAI-1 was demonstrated in mast cells of the asthmatic airway (Cho, S. H., et al. J. Immunol. 165: 3154-3161, 2000).
The human PAI-1 gene is located on chromosome 7 (q21.3-q22) and contains eight introns and nine exons distributed over about 12.3 kb (Klinger, K. W., et al. Proc. Natl. Acad. Sci. U.S.A. 84: 8548, 1987). Eight polymorphisms of the PAI-1 gene have been discovered up to now, but only a few genotypes seem to influence the synthesis and both concentration and activity of the inhibitor in plasma (Dawson, S. J., et al. J. Biol. Chem. 268: 10739, 1993; Hermans, P. W., et al. Lancet. 354: 556, 1999; Dawson, S., et al. Arteriosclero. Thromb. 11: 183, 1991; Mansfield, M. et al. Thromb. Haemost. 71: 731, 1994). The most important of these is a single guanosine insertion/deletion variation (5G or 4G) in the promoter region (4G deletion polymorphism), situated 675 bp upstream from the transcriptional start site of the PAI-1 gene (Dawson, S. J., et al. J. Biol Chem. 268: 10739, 1993; Eriksson, P., et al,. Proc. Natl. Acad. Sci USA 92: 1851, 1995). The 4G allele is correlated with increased plasma PAI-1 levels. In vitro experiments have initially shown that the 5G allele contains an additional binding site for a protein likely related to the NF-xcexaB group of transcription factors, and this binding site is absent in the 4G allele (Dawson, S. J., et al. J. Biol. Chem. 268: 10739, 1993). After stimulation with IL-1, HepG2 cells transfected with the 4G allele produce six times more PAI-1 mRNA than those with the 5G allele. These data suggest a functional role of the 4G/5G polymorphism in response to cytokines, the 4G allele being associated with enhanced gene expression (Dawson, S. J., et al. J. Biol. Chem. 268: 10739, 1993). Both alleles bind a transcriptional activator, whereas the 5G allele also binds a repressor protein to an overlapping binding site, which decreases the binding of the activator by interference due to steric hindrance. A relationship between increased PAI-1 levels in plasma and the 4G polymorphism has been described in patients with cardiovascular and metabolic diseases (Dawson, S. J., et al. J. Biol. Chem. 268: 10739, 1993; 16-20).
Treatment of bronchial asthma patients with prednisone resulted in an increase of PAI-1 activity (Banach-Wawrzenczyk, E. et al., Pol. Merkuriusz Lek 7(43): 9-11, 2000; Dziedzicko, A. et al., Pneumonol. Alergol Pol 66(3-4): 173-177, 1998). No statistical difference were found with other fibrinolysis factors after the treatment.
Bleomycin-induced lung injury was reported to result in increased PAI-1 activity levels (Olman, M. A. et al. J. Clin. Invest. 96(3): 1621-1630, 1995). In situ hybridization showed mRNA induction. The observations suggested that PAI-1 expression plays an important role in the formation and persistence of extracellular fibrin in injured lung tissue.
There exists a need for asthma treatments and chronic obstructive pulmonary discase treatments. These treatments may be able to take advantage of the observed PAI-1 activity levels.
Antagonists to plasminogen activator inhibitor type-1 (PAI-1) can be used for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Antagonists can be antibodies, peptides, proteins, nucleic acids, small organic molecules, or polymers.
The following sequence listings form part of the present specification and are include to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these sequences in combination with the detailed description of specific embodiments presented herein.
The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.
xe2x80x9cAntibodiesxe2x80x9d refers to whole antibodies and antibody fragments or molecules including antibody fragments, including, but not limited to, single chain antibodies, humanized antibodies, DEIMMUNISED(trademark) antibodies, and Fab, F(abxe2x80x2)2, VH, VL, Fd, and single or double chain Fv fragments.
xe2x80x9cPAIxe2x80x9d refers to plasminogen activator inhibitor; xe2x80x9cPAI-1xe2x80x9d refers to plasminogen activator inhibitor type-1.
xe2x80x9cTDTxe2x80x9d refers to transmission disequilibrium test.
xe2x80x9cECMxe2x80x9d refers to extracellular matrix.
xe2x80x9ctPAxe2x80x9d refers to tissue-type plasminogen activator.
xe2x80x9cuPAxe2x80x9d refers to urokinase-type plasminogen activator.
xe2x80x9cFITCxe2x80x9d refers to fluorescein-isothiocyanate.
PAI-1 is shown to be highly expressed in the airways of a murine asthma model. Additionally, the 4G allele was shown to be preferentially transmitted to asthmatic children. These results suggest a possible role of PAI-1 gene and the 4G polymorphism in the pathophysiology of asthma. Antagonists of PAI-1 can be used to reduce or eliminate asthma or chronic obstructive pulmonary disease.
The induction of the PAI-1 gene in the lung tissue of a murine asthma model was shown using both a RT-PCR and an immunofluorescence approach. Although many cell types are capable of synthesizing PAI-1 (Loskutoff, D. J., M. Sawdey, and J. Mimuro. Prog. Hemost. Thromb. 9: 87, 1995), endothelial cells may be the major source of PAI-1 under basal conditions (Yamamoto, C., et al. Thromb. Res. 74: 163, 1994). Prinsky et al. demonstrated that macrophages appeared to be the principal cell type secreting PAI-1 in lung tissue under hypoxic conditions (J. Clin. Invest. 102: 919, 1998). Mast cells have been shown to be one of the important sources of PAI-1 in the asthmatic airways (Cho, S. H., et al. J. Immunol. 165: 3154, 2000). Taken together, the main sources of increased PAI-1 levels in the lung tissue of this murine asthma model appear to be endothelial cells, macrophages, and mast cells. PAI-1 secretion was shown to be increased in the airways of the murine asthma model, suggesting that PAI-1 secretion may also be increased in the asthmatic airways. These results are consistent with the report demonstrating that the levels of PAI-1 in the BAL fluids were increased in patients with idiopathic pulmonary fibrosis (Kotani, I., et al. Thromb Res. 77: 493, 1995).
In order to investigate the potential contribution of polymorphism with the PAI-1 gene to the development of asthma nuclear families were recruited from Nottingham UK on the basis of affected sib-pairs for asthma. Using a TDT approach, preferential transmission of the 4G allele to asthmatic children was demonstrated. The trend towards increased transmission of the 4G allele was also seen with the phenotype atopy, but the number of informative families was smaller due to the high prevalence of atopy within these families and no significant effect was observed. The prevalence of the 4G allele in the Nottingham families is slightly higher than that reported in a previous UK study on healthy individuals (Hermans, P. W., et al. Lancet. 354: 556, 1999) and the prevalence of the 4G allele was also higher than that reported in previous population based studies in caucasian populations (Eriksson, P., B. et al. Proc. Natl. Acad. Sci USA 92: 1851, 1995; Westendorp, R. G., et al. Lancet. 354: 561, 1999). This could be explained either by differing prevalence of the 4G allele in different caucasian populations or by the possibility that individuals carrying the 4G allele may have been preferentially included in this study because of an association of the allele with asthma. The demonstration of increased transmission of the 4G allele in asthmatic siblings is in keeping with the observed functional effects of this allele in vitro (Dawson, S. J., et al. J. Biol. Chem. 268: 10739, 1993), with increased levels of PAI-1 in the plasma of individuals carrying this allele due to increased transcriptional activity of the gene. One would also predict that in addition to potentially contributing to the development of asthma per se, this polymorphism might be important in determining the severity of the disease and could contribute to the development of bronchial hyperresponsiveness.
These results suggests that the gene for PAI-1 may predispose to the development of asthma and contribute to the airway remodeling seen in a model of chronic asthma. Inhibitors of PAI-1 (Eitzman, D. T., et al. J. Clin. Invest. 95: 2416, 1995) can inhibit the development of asthma or alter the chronic airway remodeling which occurs in the disease.
Antagonists to PAI-1 can be used in the treatment of asthma and chronic obstructive pulmonary disease. Antagonists can be antibodies, peptides, proteins, nucleic acids, small organic molecules, or polymers. Preferably, the antagonist is an antibody. Antagonists may be prepared as a composition with a pharmaceutically acceptable carrier or diluent. Carriers and diluents refer to any and all solvents, dispersion media, antibacterial agents, antifungal agents, microcapsules, liposomes, cationic lipid carriers, isotonic and absorption delaying agents and the like which are not incompatible with the antagonist.
The antibody can be a monoclonal or polyclonal antibody. The antibody can be chemically linked to another organic or bio-molecule. Monoclonal and polyclonal antibodies may be made by any method generally known to those of skill in the art. U.S. Pat. No. 5,422,245 (issued Jun. 6, 1995) describes the production of monoclonal antibodies to plasminogen activator inhibitor.
Peptides, proteins, nucleic acids, small organic molecules, and polymers may be identified by combinatorial methods.
Known PAI-1 antagonists may be used, for example spironolactone, imidapril, angiotensin converting enzyme inhibitors (ACEI, captopril, or enalapril), angiotensin II receptor antagonist (AIIRA), or defibrotide (a polydeoxyribonucleotide).
PAI-1 antisense nucleic acid molecules may be used to reduce the levels of PAI-1 in treating asthma or chronic obstructive pulmonary disease.
An embodiment of the invention is directed towards a method to treat asthma in a mammal. The method preferably comprises selecting a mammal diagnosed with asthma, and administering to the mammal a plasminogen activator inhibitor-1 antagonist. The antagonist is preferably administered at a concentration suitable to reduce the effects of asthma. The concentration of the antagonist is preferably less than about 100 xcexcM, about 10 xcexcM, about 1 xcexcM, about 0.1 xcexcM, about 0.01 xcexcM, about 0.001 xcexcM or about 0.0001 xcexcM. The administering step can be performed by any acceptable means, including oral, inhalation, topical, IV, IP, and IM administration. The mammal can generally be any mammal susceptible to asthma, preferably is a human, a cat, a dog, a cow, a horse, a pig, or a goat, and more preferably is a human. The plasminogen activator inhibitor-1 antagonist can generally be any plasminogen activator inhibitor-1 antagonist. Preferably, the plasminogen activator inhibitor-1 antagonist is an antibody, a protein, a peptide, a polynucleotide, or a small organic molecule. The antibody can be a monoclonal antibody or a polyclonal antibody. The plasminogen activator inhibitor-1 antagonist can be spironolactone, imidapril, an angiotensin converting enzyme inhibitor, captopril, enalapril, an angiotensin II receptor antagonist, or defibrotide.
An additional embodiment of the invention is directed towards a method to treat chronic obstructive pulmonary disease in a mammal. The method preferably comprises selecting a mammal diagnosed with chronic obstructive pulmonary disease, and administering to the mammal a plasminogen activator inhibitor-1 antagonist. The antagonist is preferably administered at a concentration suitable to reduce the effects of chronic obstructive pulmonary disease. The concentration of the antagonist is preferably less than about 100 xcexcM, about 10 xcexcM, about 1 xcexcM, about 0.1 xcexcM, about 0.01 xcexcM, about 0.001 xcexcM or about 0.0001 xcexcM. The administering step can be performed by any acceptable means, including oral, inhalation, topical, IV, IP, and IM administration. The mammal can generally be any mammal susceptible to asthma, preferably is a human, a cat, a dog, a cow, a horse, a pig, or a goat, and more preferably is a human. The plasminogen activator inhibitor-1 antagonist can generally be any plasminogen activator inhibitor-1 antagonist. Preferably, the plasminogen activator inhibitor-1 antagonist is an antibody, a protein, a peptide, a polynucleotide, or a small organic molecule. The antibody can be a monoclonal antibody or a polyclonal antibody. The plasminogen activator inhibitor-1 antagonist can be spironolactone, imidapril, an angiotensin converting enzyme inhibitor, captopril, enalapril, an angiotensin II receptor antagonist, or defibrotide.
An additional embodiment of the invention is directed towards the use of compounds which change the concentration of upstream regulators or downstream effector molecules of PAI-1, in treating or preventing asthma or chronic obstructive pulmonary disease. The method can comprise selecting a mammal diagnosed with asthma or chronic obstructive pulmonary disease, and administering to the mammal one or more compounds. The compounds can comprise urokinase, tissue plasminogen activator, vitronectin, plasminogen, plasmin, matrix metalloproteinases, or tissue inhibitors of metalloproteinases. The concentration of compound is preferably less than about 100 xcexcM, about 10 xcexcM, about 1 xcexcM, about 0.1 xcexcM, about 0.01 xcexcM, about 0.001 xcexcM or about 0.0001 xcexcM. The administering step can be performed by any acceptable means, including oral, inhalation, topical, IV, IP, and IM administration. The mammal can generally be any mammal susceptible to asthma or chronic obstructive pulmonary disease, preferably is a human, a cat, a dog, a cow, a horse, a pig, or a goat, and more preferably is a human.
An additional embodiment of the invention is directed towards methods for the prevention of asthma and/or chronic obstructive pulmonary disease. The methods can comprise selecting a mammal, and administering to the mammal a plasminogen activator inhibitor-1 antagonist. The antagonist is preferably administered at a concentration suitable to reduce the occurrence or effects of asthma or chronic obstructive pulmonary disease relative to a mammal which did not receive the administration. The concentration of the antagonist is preferably less than about 100 xcexcM, about 10 xcexcM, about 1 xcexcM, about 0.1 xcexcM, about 0.01 xcexcM, about 0.001 xcexcM or about 0.0001 xcexcM. The administering step can be performed by any acceptable means, including oral, inhalation, topical, IV, IP, and IM administration. The mammal can generally be any mammal susceptible to asthma or chronic obstructive pulmonary disease, preferably is a human, a cat, a dog, a cow, a horse, a pig, or a goat, and more preferably is a human. The plasminogen activator inhibitor-1 antagonist can generally be any plasminogen activator inhibitor-1 antagonist. Preferably, the plasminogen activator inhibitor-1 antagonist is an antibody, a protein, a peptide, a polynucleotide, or a small organic molecule. The antibody can be a monoclonal antibody or a polyclonal antibody. The plasminogen activator inhibitor-1 antagonist can be spironolactone, imidapril, an angiotensin converting enzyme inhibitor, captopril, enalapril, an angiotensin II receptor antagonist, or defibrotide.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.